Intracellular trafficking of membrane-bound receptors plays a critical role in receptor processing and signal regulation. In yeast, a retromer complex consisting of five proteins (Vps5p, Vps17p. Vps26p, Vps29p, and Vps35p) directs trafficking of the membrane-bound hydrolase receptor Vps10p between the vacuole and trans-Golgi network. Sorting nexins 1 and 2 (SNX1 and SNX2), the human orthologs of Vps5p and members of a putative mammalian retromer complex, have likewise been attributed a role in protein trafficking in mammalian cells. SNX1 has been implicated in the normal trafficking of the epidermal growth factor receptor (EGFR) and the thrombin receptor PARI to lysosomes for degradation in mammalian cells, a critical step in receptor down-regulation. Null mutations in Snx1 and Snr2 were recently generated in mice in order to assess the in vivo roles of the proteins. While the singly-deficient animals are outwardly normal, the doubly-deficient embryos die by embryonic day 10.5. Furthermore, a null mutation in the mouse ortholog of the retromer component Vps26, Hbeta58, results in embryonic lethality by E9.5. The similarity of these phenotypes suggests a potentially critical role for a mammalian retromer complex in embryonic development. The broad objective of this proposal is to define the roles of SNX1, SNX2, and Hbeta58 in receptor trafficking and signaling using fibroblasts derived from genetically modified embryos. The cells will be analyzed for morphological abnormalities, receptor mis-trafficking, and dysregulated receptor signaling that would contribute to an understanding of Snx1-/-;Snx2-/- and Hbeta58-/- embryonic lethality. The proposed experiments address the roles of three components of a putative mammalian retromer complex in growth factor receptor trafficking and signaling, the misregulation of which is associated with tumorigenesis, and in embryonic development.